1. Field of the Invention
This invention relates to an improved optical method and sensor for polyhydroxy substituted organic molecules that measure the concentration of these molecules in aqueous media. In particular, the method and sensor monitor the concentration of sugars, i.e. glucose or fructose, in aqueous solution in vivo. The determination of glucose in physiological fluids in vivo is of particular importance. The sensing device is implanted in a human being. Some of the novel components of the optical method and device are also considered to be inventions in their own right.
2. Description of Related Art
There has been an ongoing effort over many years to use fluorescence techniques to measure polyhydroxyl compound (e.g. glucose) concentrations in body fluids. Although the term xe2x80x9cglucosexe2x80x9d is used herein below, it is to be understood that the concentration of most polyhydroxyl-containing organic compounds (carbohydrates, 1,2-diols, 1,3-diols and the like) in a solution are determined. But in spite of the intense effort, no practical system has been developed and commercialized for in vivo monitoring. Several attempts have been made to detect glucose by fluorescence using dyes to which a boronic acid group has been attached. Boronic acids are known to bind sugars reversibly. When the boronic acid functional dye binds to a sugar, the properties of the dye are affected. These changes have been used in the past to measure sugar concentration.
One use of this approach to a glucose sensor was reported by Russell U.S. Pat. No. 5,137,833 (See also Russell and Zepp, U.S. Pat. No. 5,512,246) which disclosed the use of a boronic acid functionalized dye that binds to glucose and generate a signal dependent on glucose concentration. James et al U.S. Pat. No. 5,503,770 used the same principle but combined a fluorescent dye, an amine quenching functionality, and a boronic acid in a single complex moiety, the fluorescence emission from which varies with extent of glucose binding. Van Antwerp et al U.S. Pat. Nos. 6,002,954 and 6,011,984 combined features of the previously cited references and also taught fabrication of a device that is purported to be implantable.
Patents of interest include but are not limited to:
Russell, U.S. Pat. No. 5,137,833 (1992)
James et al, U.S. Pat. No. 5,503,770 (1996)
Russell and Zepp, U.S. Pat. No. 5,512,246 (1996)
Van Antwerp et al, U.S. Pat. No. 6,002,954 (1999)
Van Antwerp and Mastrototaro, U.S. Pat. No. 6,011,984 (2000)
Related U.S. patents of interest include:
Wolfbeis et al, U.S. Pat. No. 4,586,518 (1986)
Gallop and Paz, U.S. Pat. No. 4,659,817 (1989)
Yafuso and Hui, U.S. Pat. No. 4,798,738 (1989)
Yafuso and Hui, U.S. Pat. No. 4,886,338 (1989)
Saaski et al, U.S. Pat. No. 5,039,491 (1991)
Lanier et al, U.S. Pat. No. 5,114,676 (1992)
Wolfbeis et al, U.S. Pat. No. 5,232,858 (1993)
Colvin, U.S. Pat. No. 5,517,313 (1996)
Sundrehagen et al, U.S. Pat. No. 5,631,364 (1997)
James et al, U.S. Pat. No. 5,763,238 (1998)
Siegmund et al, U.S. Pat. No. 5,711,915 (1998)
Bamard and Rouilly, U.S. Pat. No. 5,852,126 (1998)
Colvin, U.S. Pat. No. 5,894,351 (1999)
Alder et al, U.S. Pat. No. 5,922,612 (1999)
Arnold et al, U.S. Pat. No. 6,063,637 (2000)
Song et al, U.S. Pat. No. 6,046,312 (2000)
Kimball et al, U.S. Pat. No. 6,139,799 (2000)
Clark et al., U.S. Pat. No. 6,040,194 (2000)
Related articles and publications of interest include:
Yoon and Czarnik, J. Amer. Chem. Soc. (1992) 114, 5874-5875
James, Linnane, and Shinkai, Chem.Commun. (1996), 281-288
Suenaga et al, Tetrahedron Letters (1995), 36, 4825-4828
Eggert et al, J.Org.Chem. (1999), 64, 3846-3852
Wolfbeis et al, Analytica Chimica Acta (1995), 304, 165-170
Wang et al, Organic Letters (1999), 1, 1209-1212
Chen et al, Proc. Nat. Acad. Sci. (1999), 96, 12287-12292
P. D. Hale et al, Analytica Chimica Acta (1999), 248, 155-161
A. E. Colvin, Jr. et al, Johns Hopkins Technical Digest, Vol.12, #17, p. 378 (1996)
Murakami et al, Chem. Letters (Japan) (2000), (8), p. 940-941.
References of a general nature include:
A. W. Czarnik (ed), Fluorescent Chemosensors for Ion and Molecule Recognition, ACS Washington, D.C. 1992.
F. W. Scheller et al (eds), Frontiers in Biosensorics I Fundamental Aspects, Birkhxc3xa4user Verlag, Basel 1997.
J. R. Lakowicz, Principles of Fluorescence Spectroscopy 2nd ed. Kluwer Academics/Plenum Publishers, New York, N.Y. (1999).
Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals 6th ed. Molecular Probes Inc. Eugene, Oreg. (1996).
All patents, articles, references, standards and the like cited in this application are incorporated herein by reference in their entirety.
All of these prior art sensors are deficient in one or more aspects, such as operability under physiological conditions, stability of operation, simplicity of design, reliability, implantability, and sensitivity. The present invention overcomes these deficiencies.
This present invention concerns an optical method and an optical device for determining in vivo the concentrations of polyhydroxyl compounds, especially sugars such as glucose or fructose, in physiological media. These compounds, the analytes, are in a system with a fluorescence sensing device comprised of a light source, a detector, and the active components including a fluorophore (fluorescent dye), a quencher and an optional polymer matrix. Some components are inventions in their own right. When excited by light of appropriate wave length, the fluorophore emits light (fluoresces). The intensity of the light is dependent on the extent of quenching. The fluorophore and quencher are preferably independent entities, optionally they are immobilized in or covalently attached to a polymeric matrix which is permeable to or in contact with the compounds of interest to be detected and quantified.
In one aspect, the present invention comprises a class of fluorescence quenching compounds that are responsive to the presence of polyhydroxyl compounds such as glucose in aqueous media at or near physiological pH. In other words, the quenching efficiency is controlled by the concentration of these compounds in the medium. The quencher is comprised of a viologen substituted with at least one boronic acid group wherein the adduct is immobilized in or covalently bonded to a polymer. The quencher, dye and polymer may also be covalently bonded to each other.
The combination of boronic acid and viologen, and the resultant effect on viologen properties are important embodiments of the present invention.
In another aspect, the present invention is a class of polymeric fluorescent dyes which are susceptible to quenching by the viologen/boronic acid adduct. Useful dyes include pyranine derivatives (e.g. hydroxypyrene trisulfonamide derivatives and the like). (See FIGS. 1 and 2),
In one embodiment, the dye is comprised of a hydroxypyrene trisulfonamide moiety bonded to a polymer. Converting sulfonic acid groups to sulfonamide groups shifts the pKa of pyranine into a range more suitable for measurement at physiological pH. This conversion also shifts the absorbance of the dye to longer wavelengths thereby allowing it to be more efficiently excited by light from a blue LED which is a preferred light source for an implanted sensor. These derivatives are typically prepared by reacting a trisulfonyl chloride intermediate with 1) a polyamine, 2) an amine functional ethylenically unsaturated monomer which adduct is subsequently polymerized, 3) or an amine functional polymer. Preferably, the dye is a fully substituted trisulfonamide containing no residual sulfonic acid groups.
In another aspect, the present invention is a composite water-compatible polymer matrix, preferably a hydrogel, which comprises the dye and quencher moieties. The matrix is a water-swellable copolymer, preferably crosslinked, to which the dye and quencher moieties are covalently bonded. More preferably, the matrix is an interpenetrating polymer network (IPN) with the dye incorporated in one polymer network and the quencher in the other polymer network. Most preferably the matrix is a semi-IPN wherein the dye component is a high molecular weight water-soluble or dispersible polymer trapped in a crosslinked network comprised of quencher monomer and suitable hydrophilic comonomers. Optionally, the quencher may be in the water-compatible or dispersible component and the dye within the network. Further both dye and quencher may be separately incorporated in water-soluble or dispersible polymers wherein dye and quencher are both trapped in an inert polymer matrix. Optionally, the components are separated from the analyte solution by a membrane which is impermeable to the components, but permeable to the analyte. Optionally, the matrix is molecularly imprinted to favor association between dye and quencher, and to enhance selectivity for specific sugars, e.g. glucose, over other polyhydroxy compounds.
In another aspect, the present invention concerns a device for measuring the concentration of glucose in vivo by means of an optical sensor. The specific device is comprised of a visible light source, preferably a blue LED light source, a photodetector, a light conduit such as an optical fiber assembly, and a water-insoluble polymer matrix comprised of a fluorophore susceptible to quenching by a viologen, a viologen/boronic acid quencher, and a glucose permeable polymer, wherein the matrix is in contact with said conduit and with the medium containing the analyte.
In another embodiment the present invention relates to an optical method for the in vivo detection between about 430 and 800 nm of polyhydroxyl-substituted organic molecules as the analyte in a physiological fluid, at pH of about 7.3 to 7.5, which method comprises:
A. obtaining a fluorophore dye D, which is compatible with the analyte solution, wherein D is selected from:
a) D1 which is a fluorophore dye having the properties of:
i. A fluorophore,
ii. An excitation in the range greater than 430 nm and less than 800 nm,
iii. Resistant to photobleaching under the conditions of analysis,
iv. A Stokes shift of about or greater than 30 nm,
v. Compatibility with said analyte solution, and wherein
vi. Said Dye D1 is quenched by methyl viologen to produce an experimentally determined apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50,
wherein the fluorophore dye D1 is a discrete compound having a molecular weight of 1,000 daltons or greater or is a pendant group or chain unit in a water soluble or dispersible polymer having a molecular weight greater than about 10,000 daltons, optionally said polymer is non-covalently associated with a water insoluble polymer matrix M1 and is physically immobilized within said polymer matrix M1; and wherein said polymer matrix M1 is permeable to or in contact with said analyte solution;
(b) D2 is a fluorophore dye having the properties of:
i. A fluorophore,
ii. An excitation in the range greater than 430 nm and less than 800 nm,
iii. A Stokes shift of about or greater than 30 nm,
iv. Resistant to photobleaching under the conditions of analyses,
v. Compatibility in the analyte solution, and wherein
vi. Said Dye D2 is quenched by methyl viologen to produce an apparent Stern-Volmer quenching constant (Ksv) greater than or equal to 50, wherein D2 is covalently bonded to a water-insoluble polymer matrix M1 wherein said polymer matrix M1 is permeable to or in contact with said analyte; wherein said fluorophore dye D2 is a part of the structure:
M1xe2x80x94L1xe2x80x94D2
wherein:
M1 is said polymer matrix, xe2x88x9dL1 is a hydrolytically stable divalent linking group selected from a direct bond and a lower alkylene having 1 to 8 carbon atoms, optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, thiourea or amine, and
D2 is said fluorophore dye which is covalently bonded to said polymer matrix M1, with the proviso that D2 which is polyfunctional is bonded to matrix M1 at one, two or three sites;
B. combining with an analyte solution-compatible boronic acid-containing quencher molecule Q, wherein Q is a conjugated nitrogen-containing heterocyclic aromatic bis-onium salt selected from:
(i) Q1 which is a pendant group or a chain unit in a water-soluble or water-dispersible polymer having a molecular weight greater than 10,000 daltons and said polymer optionally is non-covalently associated with the optional polymer matrix M1 when present and immobilized within said polymer matrix M1, wherein Q1 is a compound having the properties of:
compatibility in said analyte solution,
produces a detectable change in the emission of the dye in the presence of said analyte, or
(ii) Q2 which is a structure having the properties of:
compatibility in said analyte solution
produces a detectable chance in the emission of the dye in the presence of said analyte,
wherein Q2 is covalently bonded by a linking group L2 to M1 or to a second water-insoluble polymer matrix M2 producing M2xe2x80x94L2xe2x80x94Q2 wherein L2 is selected from a direct bond and a lower alkylene having 1 to 8 carbon atoms, optionally terminated with or including one or more divalent connecting groups selected from sulfonamide, amide, ester, ether, sulfide, sulfone, phenylene, urethane, urea, thiourea or amine,
wherein said quencher Q1 or Q2 is mixed at a molecular level with said fluorophore dye D1 or D2, and with the proviso that Q2 being polyfunctional is linked to the matrix M2 at one or two sites,
C. contacting a physiological fluid which contains the analyte, a dye and a quencher in vivo with an excitation light source coupled with a detector;
D. producing a detectable and quantifiable signal in the range of about 430 to 800 nm; and
E. determining the concentration of said polyhydroxyl-substituted analyte in said physiological fluid.
In another aspect of the method, the Dye D1 is selected from pyranine derivatives having the structure of: 
where R1, R2 and R3 are each xe2x80x94NHxe2x80x94CH2xe2x80x94CH2(xe2x80x94Oxe2x80x94CH2xe2x80x94CH2)nxe2x80x94X1,
wherein X1 is selected from xe2x80x94OH, xe2x80x94OCH3, xe2x80x94CO2H, xe2x80x94CONH2, xe2x80x94SO3H, or xe2x80x94NH2; and n is between about 70 and 10,000, and preferably between 100 and 1,000.
In another aspect of the method, the Dye D2 is prepared from pyranine derivatives having the structure of: 
or from a dye monomer selected from the group consisting of: 
where R4=xe2x80x94H
and
R5 is selected from: xe2x80x94R6xe2x80x94NHxe2x80x94(Cxe2x95x90O)xe2x80x94(Cxe2x95x90CH2)xe2x80x94R7, xe2x80x94R6xe2x80x94Oxe2x80x94(Cxe2x95x90O)xe2x80x94(Cxe2x95x90CH2)xe2x80x94R7, xe2x80x94CH2xe2x80x94C6H4xe2x80x94CHxe2x95x90CH2, or xe2x80x94CH2xe2x80x94CHxe2x95x90CH2 
where in R6 is a lower alkylene of 2 to 6 carbons and R7=xe2x80x94H, or xe2x80x94CH3 
where Z is a blocking group that is removed by hydrolysis selected from:
xe2x80x94(Cxe2x95x90O)xe2x80x94R8xe2x80x94Y
where R8 is a lower alkylene of 1 to 4 carbon atoms and
Y is selected from xe2x80x94H, xe2x80x94OH, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94(Cxe2x95x90O)xe2x80x94NHxe2x80x94R9, or xe2x80x94CO2xe2x80x94R9 
where R9 is a lower alkylene of 1 to 4 carbon atoms.
In another aspect of the method, the quencher Q1 or Q2 is prepared from a quencher precursor selected from the group consisting of: 
where (V)2+ is a nitrogen containing conjugated heterocyclic aromatic group selected from isomers of dipyridyls, dipyridyl ethylenes, dipyridyl phenylenes, phenanthrolines, or diazafluorenes; wherein the two nitrogen atoms are each in a different aromatic ring and the nitrogens are in all positions capable of forming an onium salt and where Z1 or Z2 is either a polymerizable ethylenically unsaturated group selected from:
(i) xe2x80x94R10xe2x80x94CO2xe2x80x94C(R11)xe2x95x90CH2, xe2x80x94R10xe2x80x94NHxe2x80x94(Cxe2x95x90O)xe2x80x94C(R2)xe2x95x90CH2, or xe2x80x94CH2xe2x80x94C6H4xe2x80x94CHxe2x95x90CH2,
here R10 is a lower alkylene or hydroxyalkylene of 2 to 6 carbon atoms and where R11=xe2x80x94H or xe2x80x94CH3; or
(ii) a coupling group selected from: xe2x80x94R12xe2x80x94Y1 
where R12 is xe2x80x94CH2C6H4xe2x80x94 or alkylene of 2 to 6 carbon atoms and
Y is xe2x80x94OH, xe2x80x94SH, xe2x80x94CO2H, or xe2x80x94NH2. Q1 is a pendant group or chain unit in a water-soluble or dispersible polymer (linear or branched). The polymer matrix M2xe2x80x94L2xe2x80x94Q2 is preferably a water-insoluble (crosslinked) network polymer.
For the dye D, note that D1 and D2 are defined with the proviso that the dye D1 and D2 do not include a diazo linkage xe2x80x94Nxe2x95x90Nxe2x80x94.
For the quencher Q, Q1 and Q2 are defined with the proviso that the quencher Q1 and Q2 do not include a diazo linkage xe2x80x94Nxe2x95x90Nxe2x80x94.
For the in vivo applications, described herein, the ortho-benzylboronic acid derivatives are excluded.